Monooxygenase mutants for biosynthesis of 2,6-bis(hydroxymethyl)pyridine and a method for preparation of 2,6-bis(hydroxymethyl)pyridine using the said monooxygenase mutants

Abstract

The present invention relates to the provision of an enzymatic method for the preparation of 2,6-bis(hydroxy methyl)pyridine starting from 2,6-lutidine using a mutated xylene monooxygenase enzyme, termed ppXMO, comprising a xylM subunit and a xylA subunit from Pseudomonas putida, wherein said mutated enzymes harbor an amino acid exchange at position 116 of the amino acid sequence of XylM component. The essence of the invention is that the methionine (M) at this position is replaced with an aminoacid selected in the group consisting of asparagine (N), lysine (K), arginine (R) and glycine (G), which surprisingly results in a direct methyl hydroxylation of 6-methyl-2-pyridine methanol resulting in improved overall process yield, less side products are produced, avoidance of toxic reaction intermediates and minimizing the need for involvement of endogenous reductase enzymes as well as NADPH and its regeneration. Other enzymes related to XylM of P. putida harbouring the same amino acid exchange at the highly conserved region around position 116 or its equivalent also exhibit similar improved characteristics.

1 . An enzyme having a sequence SEQ ID NO: 1 or at least 50% homology on the amino acid level to the said sequence, said homology ensuring enzymatic activity of the said enzyme, said protein having a mutation at position 116 or an equivalent position, wherein the mutation is a replacement of methionine (M) or tryptophan (W) by a different amino acid. 
     
     
         2 . The enzyme according to  claim 1 , wherein the M or W at position 116 or equivalent is replaced with an amino acid selected in the group consisting of G, N, R, or K, preferably with G. 
     
     
         3 . The enzyme according to  claim 1  having further mutations and/or deletions. 
     
     
         4 . The enzyme according to  claim 1 , wherein the enzyme is a:
 XylMA enzyme of  Pseudomonas putida , or   XylMA-like enzyme of
   Alteromonas macleodii , or 
   Tepidiphilus succinatimandens , or 
   Novosphingobium kunmingense , or 
   Hyphomonas oceanitis , or 
   Sphingobium  sp. 32-64-5 or 
   Halioxenophilus aromaticivorans  or 
   a XylMA-like enzyme with more than 50% sequence identity to SEQ ID NO: 1 on the amino acid level.   
     
     
         5 . A nucleic acid encoding the enzyme according  claim 1 . 
     
     
         6 . An expression vector comprising the nucleic acid according to  claim 5 . 
     
     
         7 . A host cell with the nucleic acid and/or the expression vector expressing the enzyme according to  claim 1 . 
     
     
         8 . The host cell according to  claim 7 , wherein the host cell is a microbial cell, preferably a bacterial cell. 
     
     
         9 . The host cell according to  claim 8 , wherein the host cell is a cell of  Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putida, Rhodobacter sphaeroides, Streptomyces  spp,  Propionibacterium shermanii, Ketogulonigenium vulgare, Acinetobacter baylyi, Halomonas  bluephagenesis, most preferably an  E. coli  cell. 
     
     
         10 . Use of the enzyme, the nucleic acid, and/or the host cell according to  claim 1, claim 5, or claim 7  in a process for the transformation of 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I. 
     
     
         11 . A process for the transformation of 2,6-lutidine II to 2,6-bis(hydroxymethyl)pyridine I, 
       
         
           
           
               
               
           
         
         wherein the transformation is performed in the presence of enzymes, characterized in that the enzyme or the host cell according to  claim 1 or claim 7  is used. 
       
     
     
         12 . The process according to  claim 11  wherein the feeding rate of 2,6-lutidine II in the reaction medium is adjusted such that the concentration of 2,6-lutidine II does not exceed the value of 1 g/L, preferably 0.1 g/L, and more preferably 0.02 g/L in a reaction medium, and wherein the feeding rate of 2,6-lutidine II in the reaction medium is adjusted such that the concentration of 2,6-lutidine II does not fall below the value of 10 mg/L, preferably 0.1 mg/L, more preferably 0.01 mg/L. 
     
     
         13 . A process according to  claim 8 or 9 , wherein a dehydrogenase is used, wherein the dehydrogenase is preferably co-expressed in the microbial host. 
     
     
         14 . A process according to  claim 13 , wherein the dehydrogenase is NADH dependent, NADP dependent, NADPH dependent or GDH dependent, wherein the dehydrogenase is preferably selected from the list of the AKR from  Kluyveromyces lactis , XylB from  Acinetobacter baylyi  ADP1, and AFPDH from  Candida maris.    
     
     
         15 . A process according to  claim 13 , wherein a NADH regeneration system, a NADP regeneration system, a NADPH regeneration system or a GDH regeneration system is co-expressed in the microbial host, wherein the NADH regeneration system is preferably a formate dehydrogenase-based system, wherein the NADH regeneration system is preferably comprised of a metal-independent formate dehydrogenase active on NAD+ species and of bacterial or fungal origin. 
     
     
         16 . A process according to  claim 15 , wherein the feeding rate of formate is such that the concentration of formate in the reaction medium does not exceed the value of 150 mM, preferably 100 mM, more preferably 50 mM, and wherein the feeding rate of formate is such that the concentration of formate does not fall below the value of 50 mM, preferably 25 mM, more preferably 5 mM, in the reaction medium. 
     
     
         17 . Use of product of the process according to  claim 11  in preparation of other compounds, diagnostic complexes, most preferably 2-[3,9-bis[1-carboxylato-4-(2,3-dihydroxypropylamino)-4-oxobutyl]-3,6,9,15-tetraza bicyclo[9.3.1]pentadeca-1 (15),11,13-trien-6-yl]-5-(2,3-dihydroxypropylamino)-5-oxopentanoate;gadolinium(3+), 2,6-Bis(chloromethyl)pyridine, 2,6-Bis(bromomethyl)pyridine, 2,6-Bis(mesyloxymethyl)pyridine and 2,6-Bis(tosyloxymethyl)pyridine. 
     
     
         18 . A process for preparation of pyridine-based tetra-aza heterocycles, preferably 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene, i.e., pyclen, comprising the method and/or products of the method according to  claim 11 . 
     
     
         19 . A process for the preparation of various diagnostic complexes or other compounds comprising the method and/or products of the method according to  claim 11 . 
     
     
         20 . The process according to  claim 19 , wherein the process is preparation of 2-[3,9-bis[1-carboxylato-4-(2,3-dihydroxypropylamino)-4-oxobutyl]-3,6,9,15-tetraza bicyclo[9.3.1]pentadeca-1(15),11,13-trien-6-yl]-5-(2,3-dihydroxypropylamino)-5-oxopentanoate;gadolinium (3+), i.e., gadopiclenol. 
     
     
         21 . A process according to  claim 10 , wherein a dehydrogenase is used, wherein the dehydrogenase is preferably co-expressed in the microbial host. 
     
     
         22 . A process according to  claim 11 , wherein a dehydrogenase is used, wherein the dehydrogenase is preferably co-expressed in the microbial host. 
     
     
         23 . A process according to  claim 12 , wherein a dehydrogenase is used, wherein the dehydrogenase is preferably co-expressed in the microbial host.